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Characterization of TaNAC-S gene in Australian wheat cultivars in relation to senescence and nitrogen stress response

Sultana, Nigarin (2020) Characterization of TaNAC-S gene in Australian wheat cultivars in relation to senescence and nitrogen stress response. PhD thesis, Murdoch University.

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TaNAC-S transcription factor (TF) was previously found to have effects on senescence and grain protein content (GPC) in wheat. However, to date, its genetic architecture, association with other genes and the underlying regulatory mechanism is still unclear, making it a challenging task to utilize its variation in wheat breeding. In the current study, molecular characterization of TaNAC-S gene was conducted in 48 Australian wheat cultivars (Triticum aestivum) with the aim of improving GPC and yield in Australian wheat cultivars. Moreover, to understand the molecular function of TaNAC-S in nitrogen metabolism and how it regulates senescence, gene expression analysis was carried out using both RNA-seq and qRT-PCR using Triticum aestivum cv. Mace, Spitfire and Volcani that differ in nitrogen use efficiency (NUE) and length of maturity. As results, one copy of TaNAC-S gene was identified in each of the group 7 chromosomes (A, B and D) and mapped to the long arms of chromosomes. The coding sequences of the genes on 7A, 7B, and 7D are 870 bp, 879 bp, and 876 bp in length, respectively, and encode proteins in size of 289, 292 and 291 residues, respectively.

Comparing the individual sequences in the selected cultivars, two alleles each were identified for TaNAC-S-7A and TaNAC-S-7B. No allelic variation was found for TaNACS-7D. Analysis of the translated protein sequences revealed no amino acid changes in TaNAC-S-7A alleles, whereas nucleotide changes in TaNAC-S-7B resulted in amino acid changes from threonine to methionine and leucine to valine. Moreover, gene sequences analysis of 1000 bp upstream of the TaNAC-S-7A promoter regions revealed a 36 bp insertion in 142 bp upstream of the start codon, while no significant difference was detected in the promoter sequences between TaNAC-S-7B alleles. TaNAC-S-7D promoter sequences were also found to be non-polymorphic across all cultivars. Further promoter motif analysis revealed the presence of multiple circadian, ethylene, NAC-ORE1, NAC-019, WRKY, BHLH, DOF and VRN1 transcription binding site motifs in 5´UTR region indicated their possible regulatory effects on TaNAC-S. Using phenotype data from glasshouse and field trial with double haploid (DH) Mace-Spitfire population, TaNAC-S-7A1 was significantly found to be associated with high chlorophyll content, flowering days, grain yield (GY) whereas TaNAC-S-7B2 was associated with higher GPC but not significantly. Transcriptome data analysis and confirmation with quantitative RT-PCR disclosed that TaNAC-S expression was higher in second leaf than flag in leaf whereas no expression found in grain tissue. Moreover, the TaNAC-S-7B expression was different from that of TANAC-S-7A and TaNAC-S-7D with significant expressional variation between the haplotypes.

Gene ontology and KEGG analysis revealed that nitrogen stress has significant down-regulatory effects on photosynthesis and light harvesting, carbohydrate metabolic process, shoot system development and stress response whereas profound up-regulatory effects on DNA topological changes, sulfate assimilation and transmembrane transport. Using gene network modelling and correlation analysis, several genes were identified that showed similar expression patterns (increased or decreased) during senescence as well as nitrogen stress, indicating importance of those genes in nitrogen stress dependent senescence. TaNAC-S was identified as one of the transcription factor genes, differentially expressed between low and high nitrogen treatments as well as during senescence with significant variation across the cultivars. Some NAC family genes showed similar gene expression (expression decrease during senescence and nitrogen stress) patterns with TaNAC-S, including TaNAC-35-2A, TaNAC-34-2A, TaNAC-29-2A and JUB1 gene homologs on chromosome 5 groups. Genes with opposite expression patterns (expression decrease during senescence and nitrogen stress) include orthologous genes of positive senescence regulators AtNAP, ORE1, ORS1 and NAM-2 in particular TaNAC-23-2A, TaNAC-9-2B, Ta-NAMB2-2B, Ta-NAMD2-2D, TaNAC-23-2A. Also, close interacting partners of TaNAC-S-7A and TaNAC-S-7D genes in gene networking analysis had association with early or late flowering control, shoot branching, and senescence while nitrogen metabolism related genes were associated with TaNAC-S-7B. Using the promoter sequence analysis, expression profile, and gene network analysis, this study suggested the possible mechanism of TaNAC-S gene function based on strigolactone-brassinosteroid hormone perception. TaNAC-S has positive association with GLK, rsbQ, SerRC whereas negative association with SQD1, CHL2, ORE1, NAC019. Among those targets that can regulate TaNAC-S, the downregulatory effect of ORE1 and NAC019 on TaNAC-S-7A1 and TaNAC-S7A2 expression were confirmed by Dual Luciferase Assay. Further functional characterization is underway to establish the interaction of GLK and PIF with TaNAC-S.

The current study identified alleles of TaNAC-S with potential breeding value in improving wheat nitrogen use efficiency. Besides, several candidate genes were also identified that can play important role in senescence regulation and nitrogen stress tolerance. The gene variation and allelic effects of TaNAC-S associated genes and the candidate genes need to be further explored to discover novel genetic sources for wheat breeding.

Keywords: Wheat (Triticum aestivum L.), Senescence, Nitrogen use efficiency (NUE), Transcription factor (TF), Grain protein content (GPC), Grain yield (GY), Wheat.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Agricultural Sciences
Supervisor(s): Ma, Wujun, Juhász, Angela, Islam, Shahidul and She, Maoyun
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